Method for synthesis of 9-cis-beta-carotene and formulations thereof

ABSTRACT

The present invention relates to a method for total chemical synthesis of 9-cis-β-carotene (9CBC), and further provides stable formulations thereof.

TECHNICAL FIELD

The present invention relates to a method for total chemical synthesisof 9-cis-3-carotene (9CBC) and to stable formulations thereof.

BACKGROUND ART

Neuro-retinal degeneration including age related macular degeneration(AMD) and retinitis pigmentosa (RP) are the leading cause of visionincapacitation and blindness worldwide. Such indications might beeffectively treated using 9CBC.

9CBC is currently obtained by isolation from a β-carotenes mixturederived from Dunaliella bardawil algae. The β-carotene mixture derivedfrom said algae contains about 50% of the 9-cis isomer; however, theexact amount of said isomer varies depending on inter alia the season,light irradiation, and temperature. Isolation of the 9-cis isomer fromthe carotenes mixture requires an extraction process that is followed byHPLC reverse phase separation phase. The product is currently availablefrom several commercial sources; however, due to the complex processinvolved, it is highly expensive.

No total synthesis of 9CBC has been reported by the date; however,extensive efforts have been made so as to develop a stereo-controlledtotal synthesis of this compound. The strategies developed for thepolyenic chain construction were based on the Wittig,Horner-Wadsworth-Emmons and related reactions; however, in all cases,mixtures of E/Z isomers were obtained and the separation of the specificisomer was quite difficult (US Patent Publication No. 20060183185).

SUMMARY OF INVENTION

In one aspect, the present invention relates to a convenient andcost-effective synthetic route for the preparation of 9CBC orderivatives thereof, which are known to have therapeutic effects in,e.g., neuro-retinal degeneration. More particularly, the inventionrelates to a method for the synthesis of 9CBC or a derivative thereof,starting from4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one,which may be synthesized from β-cyclocitral, e.g., by Reformatskyreaction of said β-cyclocitral with a compound such as ethyl4-bromo-3-methylbut-2-enoate.

The starting materials, intermediates, and final product mentioned inconnection with the synthetic process of the present invention areherein identified by the Arabic numbers 1-9 in bold, and are shown inScheme 1 illustrating the particular synthetic route exemplified.β-Cyclocitral is identified as compound 1; the lactone4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-oneand the corresponding lactol4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-olare identified as compounds 3 and 4, respectively; and 9CBC isidentified as compound 9.

More specifically, the invention relates to a method for the synthesisof 9CBC or a derivative thereof, said method comprising:

-   -   (i) reducing the lactone        4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one        (compound 3) and opening the ring of the lactol obtained        (compound 4) with complete retention of the double bond        configuration, to thereby obtain        (2Z,4E)-3-methyl-5-(2,6,6-trimethyl        cyclohex-1-en-1-yl)penta-2,4-dienal (compound 5);    -   (ii) subjecting the        (2Z,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)        penta-2,4-dienal to Horner-Emmons reaction to obtain a 9-cis        retinyl ester (compound 6);    -   (iii) reducing the 9-cis retinyl ester to obtain 9-cis retinol,        i.e., vitamin A (compound 7); and    -   (iv) converting the 9-cis retinol to its triphenylphosphonium        salt (compound 8), and subjecting said triphenylphosphonium salt        to Wittig reaction with a compound of the formula A:

-   -   -   wherein R₂ is H or methyl; X is (—C═C—C—)_(n) optionally            substituted with one or more methyl groups; and n is an            integer of 0-16, in the presence of a strong base, to            thereby obtain said 9CBC or derivative thereof.

In a particular embodiment, the triphenylphosphonium salt of 9-cisretinol is subjected, in step (iv), to Wittig reaction with all-transretinal (representing a compound of the formula A) to obtain 9CBC.

The4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one(compound 3) used as a starting material in the method of the inventionmay be synthesized by any suitable process. In particular embodiments,compound 3 is prepared from β-cyclocitral (compound 1), by reacting witha compound of the formula B:

wherein R₁ is (C₁-C₈)alkyl or (C₆-C₁₀)aryl, in the presence of metalliczinc (Zn), in a Reformatsky reaction.

In another aspect, the present invention relates to 9CBC or a derivativethereof, synthesized by the method disclosed herein, wherein saidderivative is of the formula C:

wherein R₂ is H or methyl; X is (—C═C—C—)_(n) optionally substitutedwith one or more methyl groups; and n is an integer of 0-16.

Initial stability tests indicated that synthetic 9CBC is sensitive toboth oxygen and UV degradation due to the highly conjugated nature ofthe carbon chain, resulting in full decomposition in less than 24 hours.Attempt to stabilize 9CBC by the addition of antioxidant(s), assuggested in the prior art, were unsuccessful, as such a composition hasquite rapidly degraded. As has been surprisingly found and shown herein,a 9CBC formulation comprising, as non-active ingredients, a viscosityenhancing agent such as a lecithin, e.g., a soybean lecithin, a mediumchain triglyceride such as miglyol, or a polysorbate such as polysorbate20 and polysorbate 40; as well as one or more antioxidants, are highlystable for a long period of time.

In a further aspect, the present invention thus provides a formulation,more particularly a pharmaceutical formulation/composition, comprisingan active agent selected from 9CBC or a derivative thereof of theformula C as defined above, a thickening- or solidifying-, i.e.,viscosity enhancing, agent, and an antioxidant. Such pharmaceuticalformulations are useful for treatment of any medical indication in whichadministration of 9CBC or a derivative thereof as defined above isuseful, e.g., neuro-retinal degeneration.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows HPLC reversed phase analysis of 9CBC reaction mixture(λ=447 nm).

FIG. 2 shows HPLC analysis (λ=447 nm) of purified 9CBC (2 mL of 8:2CH₃OH:CHCl₃) with 1 μL DMSO.

DETAILED DESCRIPTION

As shown herein, a multi-steps process for the synthesis of 9CBC fromcommon available compounds was developed based on stereospecificsynthesis of 9-cis olefin intermediate via a lactone ring opening withcomplete retention of double bond configuration, followed byHorner-Emmons, ester reduction and Witting reaction to produce the finalcompound.

In one aspect, the present invention relates to a method for thesynthesis of 9CBC or a derivative thereof, as defined above, i.e.,starting from the lactone4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one(compound 3).

The term “alkyl” as used herein typically means a linear or branchedsaturated hydrocarbon radical having 1-8 carbon atoms and includes,e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,tert-butyl, n-pentyl, isoamyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl,n-octyl, and the like. Preferred are (C₁-C₆)alkyl groups, morepreferably (C₁-C₄)alkyl groups, most preferably methyl, ethyl, propyl orisopropyl. The alkyl may optionally be substituted with one or moregroups each independently selected from halogen, —COR, —COOR, —CN, —NO₂,—SR, —OR, —N(R)₂, —CON(R)₂, —SO₂R, —SO₃R or —S(═O)R, wherein R is H orunsubstituted linear or branched (C₁-C₈)alkyl.

The term “aryl” as used herein denotes an aromatic carbocyclic grouphaving 6-10 carbon atoms consisting of a single ring or condensedmultiple rings such as, but not limited to, phenyl and naphthyl. Thearyl may optionally be substituted with one or more groups eachindependently selected from halogen, —COR, —COOR, —CN, —NO₂, —SR, —OR,—N(R)₂, —CON(R)₂, —SO₂R, —SO₃R, —S(═O)R, or —(C₁-C₈)alkyl, wherein R isH or unsubstituted linear or branched (C₁-C₈)alkyl.

The term “halogen” as used herein includes fluoro, chloro, bromo, andiodo, but it is preferably fluoro, chloro or bromo.

In certain embodiments, the reduction in step (i) of the method of theinvention is carried out with diisobutylaluminum hydride (DIBAL-H).

In certain embodiments, opening the ring of the lactol in step (i) ofthe method of the invention is carried out in the presence of a strongacid. Non-limiting examples of suitable acids include HCl, HClO₄, HClO₃,HNO₃, HBr, HI, and H₂SO₄. In a particular embodiment, opening the ringof the lactol in step (i) is carried out in the presence of HCl.

In certain embodiments, the Horner-Emmons reaction in step (ii) of themethod of the invention is carried out with triethyl3-methyl-4-phosphono-2-butenoate to obtain 9-cis retinyl ethyl ester.

In certain embodiments, converting the 9-cis retinol to itstriphenylphosphonium salt in step (iv) of the method of the invention iscarried out with triphenylphosphine.

In certain embodiments, the triphenylphosphonium salt of 9-cis retinolis subjected, in step (iv) of the method of the invention, to Wittigreaction with all-trans retinal, representing the compound of theformula A, to obtain 9CBC.

4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-onefor use as a starting material in step (i) of the method of theinvention may be synthesized by any suitable process. In certainembodiments, this lactone is synthesized from β-cyclocitral (compound1), by reacting β-cyclocitral with a compound of the formula B:

wherein R₁ is (C₁-C₈)alkyl or (C₆-C₁₀)aryl, in the presence of metallicZn, in a Reformatsky reaction. In a particular such embodimentexemplified herein,4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-oneis obtained by reacting 3-cyclocitral with a compound of the formula Bwherein R₁ is ethyl, i.e., with ethyl 4-bromo-3-methylbut-2-enoate(compound 2).

In another aspect, the present invention relates to 9CBC or a derivativethereof, synthesized by the method disclosed herein, wherein saidderivative is of the formula C:

wherein R₂ is H or methyl; X is (—C═C—C—)_(n) optionally substitutedwith one or more methyl groups; and n is an integer of 0-16. The 9CBC orderivative thereof prepared by the method of the present invention maybe crude or purified to a certain level if necessary, and may thuscontain varying amounts of impurities left from the synthetic procedureas disclosed herein, i.e., traces of one or more of the intermediatesformed, or reagents added, during the procedure.

In a further aspect, the present invention provides a formulationcomprising an active agent, a thickening/solidifying, i.e., viscosityenhancing, agent, and at least one (i.e., one or more) antioxidant,wherein said active agent is 9CBC or a derivative thereof of the formulaC as defined above. Particular such formulations may further comprise apharmaceutically acceptable carrier, and are thus referred to herein aspharmaceutical formulations or pharmaceutical compositions.

The term “thickening agent”, “solidifying agent”, or “viscosityenhancing agent”, used herein interchangeably, means an agent that issolid or semi-solid in the dry form, i.e., when not dissolved in aliquid medium, capable of increasing the viscosity of a liquid withoutsubstantially changing its other physico-chemical properties, and has afair solubility in non-polar organic solvents such as pentane,cyclopentane, hexane, cyclohexane, benzene, toluene, 1,4-dioxane,chloroform, diethyl ether, and dichloromethane; polar organic solventssuch as formic acid, n-butanol, tert-butanol, isopropanol, n-propanol,ethanol, methanol, acetic acid, and dimethyl sulfoxide (DMSO); water; orbuffer solutions.

In certain embodiments, the thickening/solidifying agent comprisedwithin the formulation of the present invention comprises a lecithin ora PEGylated derivative thereof; a lecithin-like substance; a mediumchain triglyceride or a mixture thereof; a povidone, also referred to aspolyvidone or polyvinylpyrrolidone (linear formula: [C₆H₉NO]_(n), CAS:9003-3908); a polysorbate; or sorbitol.

In certain particular such embodiments, said thickening/solidifyingagent comprises a lecithin or a PEGylated derivative thereof, alecithin-based product, or a povidone. More particular such embodimentsare those wherein the viscosity enhancing agent comprises egg lecithin,soybean lecithin, or a PEGylated derivative thereof; or those whereinthe viscosity enhancing agents comprises a lecithin-based product suchas L-α-lecithin, granular (Acros Organics™, Fisher Scientific), which isa soybean lecithin concentrate consisting of more than 94%phosphatidylcholine and less than 2% triglycerides; CAS: 8002-43-5)(https://www.fishersci.com/shop/products/1-alpha-lecithin-granular-acros-organics-2/p-153301;Phospholipon® 50, Phospholipon® 75, Phospholipon® 85G or Phospholipon®90G, essentially consisting of soybean lecithin and phospholipids;Phospholipon® 80H or Phospholipon® 90H, essentially consisting ofhydrogenated soybean lecithin and phospholipids; Phospholipon® E25,Phospholipon® E35 or Phospholipon® E, essentially consisting of egg yolklecithin and phospholipids; and Phospholipon® LPC20, Phospholipon® LPC25or Phospholipon® LPC65, essentially consisting of partially hydrolyzedsoybean lecithin.

In other particular such embodiments, the thickening/solidifying agentcomprised within the formulation of the invention comprises alecithin-like substance such as egg yolk.

In further particular such embodiments, the thickening/solidifying agentcomprised within the formulation of the invention comprises a mediumchain triglyceride, i.e., a triglyceride composed of a glycerol backboneand three fatty acids, wherein two or three of the fatty acids aremedium chain fatty acids, i.e., fatty acids having an aliphatic chain of6-12 carbon atoms, or a mixture thereof. Particular medium chaintriglycerides that can be used include, without limiting, miglyol, andtriglycerides of fractionated C₈-C₁₀ coconut oil fatty acids, e.g.,miglyol 810.

In yet other particular such embodiments, the thickening/solidifyingagent comprised within the formulation of the invention comprises apolysorbate, i.e., an oily liquid emulsifier derived from ethoxylatedsorbitan esterified with fatty acids. Non-limiting examples of suchpolysorbates include polysorbate 20 (polyoxyethylene sorbitanmonolaurate; Tween® 20), polysorbate 40 (polyoxyethylene sorbitanmonopalmitate; Tween® 40), polysorbate 60 (polyoxyethylene sorbitanmonostearate; Tween® 60), and polysorbate 80 (polyoxyethylene sorbitanmonooleate; Tween® 80).

In still further particular such embodiments, the thickening/solidifyingagent comprised within the formulation of the invention comprisessorbitol ((2S,3R,4R,5R)-hexane-1,2,3,4,5,6-hexol).

In certain embodiments, the antioxidant comprised within the formulationof the present invention is a thiol such as aurothioglucose,dihydrolipoic acid, propylthiouracil, thioredoxin, glutathione (GSH),L-cysteine, N-acetylcysteine (NAC), cystine, cystamine, andthiodipropionic acid; a sulphoximine such as buthionine-sulphoximine,homo-cysteine-sulphoximine, buthionine-sulphones, and penta-, hexa- orheptathionine-sulphoximine; a metal chelator such as a α-hydroxy-fattyacid, palmitic acid, phytic acid, lactoferrin, citric acid, lactic acid,malic acid, humic acid, bile acid, bile extracts, bilirubin, biliverdin,EDTA, Na₂-EDTA, Na₂-EDTA-Ca, EGTA, and DTPA; sodium bisulfite, andsodium metabisulfite; a vitamin such as vitamin E, ascorbic acid orascorbic acid salts (Na-ascorbate, ascorbyl palmitate, Mg ascorbylphosphate, and ascorbyl acetate); a phenol such as butylhydroxytoluene(BHT), butylhydroxyanisole, ubiquinol, nordihydroguaiaretic acid, andtrihydroxybutyrophenone; a benzoate such as coniferyl benzoate; uricacid; mannose; propyl gallate; a selenium such as selenium-methionine; astilbene such as stilbene oxide and trans-stilbene oxide; a carotenoidsuch as α-carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin,astaxanthin, and lycopene; or a mixture thereof. A particular mixture ofcarotenoids that might be used is the commercially available productCarotenALL, Mixed Carotenoid Complex (Jarrow Formulas;http://www.iherb.com/Jarrow-Formulas-CarotenALL-Mixed-Carotenoid-Complex-60-Softels/153.

In particular embodiments, the antioxidant comprised within theformulation of the present invention is a phenol such as BHT,butylhydroxyanisole, ubiquinol, nordihydroguaiaretic acid, andtrihydroxybutyrophenone; a carotenoid such as α-carotene, β-carotene,β-cryptoxanthin, lutein, zeaxanthin, astaxanthin, and lycopene; or amixture thereof. More particular such embodiments are those wherein saidantioxidant is BHT, carotenoid, or a mixture thereof.

In certain embodiments, the present invention provides a formulation asdefined above, wherein the viscosity enhancing agent comprises alecithin or a PEGylated derivative thereof, a lecithin-based product, ora povidone; and the antioxidant is BHT, butylhydroxyanisole, ubiquinol,nordihydroguaiaretic acid, trihydroxybutyrophenone, α-carotene,β-carotene, β-cryptoxanthin, lutein, zeaxanthin, astaxanthin, lycopene,or a mixture thereof. In some particular such embodiments, said lecithinis egg lecithin or soybean lecithin; said lecithin-based product isL-α-lecithin, granular (Acros Organics™), Phospholipon® 50,Phospholipon® 75, Phospholipon® 85G, Phospholipon® 90G, Phospholipon®80H, Phospholipon® 90H, Phospholipon® E25, Phospholipon® E35,Phospholipon® E, Phospholipon® LPC20, Phospholipon® LPC25, orPhospholipon® LPC65; or said antioxidant is BHT, a carotenoid, or amixture thereof. In other particular such embodiments, said viscosityenhancing agent comprises egg lecithin or soybean lecithin; and saidantioxidant is BHT, α-carotene, β-carotene, β-cryptoxanthin, lutein,zeaxanthin, astaxanthin, lycopene, or a mixture thereof. Preferred suchembodiments are those comprising egg lecithin or soybean lecithin as theviscosity enhancing agent, and BHT as the antioxidant. Formulations asdefined hereinabove may comprise said viscosity enhancing agent and saidantioxidant in a weight ratio that is in a range of about 100:1 to about1:100, respectively, e.g., about 100:90, 100:80, 100:70, 100:60, 100:50,100:40, 100:30, 100:20, 100:10 100:1, 1:100, 10:100, 20:100, 30:100,40:100, 50:100, 60:100, 70:100, 80:100, or 90:100 (viscosity enhancingagent:antioxidant), respectively.

In certain embodiments, the active agent comprised within theformulation of the present invention, according to any one of theembodiments defined above, is 9CBC. In other embodiments, said activeagent is a 9CBC derivative of the formula C as defined above. Inparticular such embodiments, the formulation disclosed herein comprises9CBC, a soybean lecithin, and BHT, e.g., at a weight ratio of about1:10:5, respectively.

As shown herein, 9CBC formulations according to the present inventionare chemically stable, i.e., show almost no degradation at all of theactive agent, for weeks, when stored under inert conditions, e.g., underargon or nitrogen atmosphere. Formulations according to the inventionmay thus be stored for relatively long durations of time, e.g., for atleast 72 or 96 hours, i.e., 3 or 4 days, respectively, but preferablyfor 1, 2, 3, or 4 weeks; 1, 2, 3, 4, 5, or 6 months; or more, underinert conditions and optionally at a temperature below 0° C. (e.g., atabout or less than −20° C.), without a substantial degradation of theactive agent.

The pharmaceutical formulation of the present invention, as defined inany one of the embodiments above, is in the form of a solid, semi-solid,gel, or paste, and is thus preferably formulated for oraladministration.

The pharmaceutical formulations provided by the present invention may beprepared by conventional techniques, e.g., as described in Remington:The Science and Practice of Pharmacy, 19^(th) Ed., 1995. Theformulations can be prepared, e.g., by uniformly and intimately bringingthe active agent, i.e., 9CBC or a derivative thereof as defined herein,into association with a said viscosity enhancing agent and said at leastone antioxidant; mixing said components; and then, if necessary, shapingthe product into the desired formulation. The compositions may furtherinclude pharmaceutically acceptable carriers, fillers, diluents oradjuvants, and other inert ingredients and excipients. In oneembodiment, the pharmaceutical formulation of the present invention isformulated as particles, e.g., micro particles or nanoparticles.

Pharmaceutical formulations according to the invention, for oraladministration, may be formulated as tablets, troches, lozenges, hard orsoft capsules, or syrups or elixirs. Such formulations may be preparedaccording to any method known in the art for the manufacture ofpharmaceutical compositions and may further comprise one or morenon-toxic pharmaceutical acceptable excipients, as well as agentsselected from sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. These excipients may be, e.g., inert diluentssuch as calcium carbonate, sodium carbonate, lactose, calcium phosphate,or sodium phosphate; granulating and disintegrating agents, e.g., cornstarch or alginic acid; binding agents, e.g., starch, gelatin or acacia;and lubricating agents, e.g., magnesium stearate, stearic acid, or talc.Oral compositions in the form of tablets may be either uncoated orcoated utilizing known techniques to delay disintegration and absorptionin the gastrointestinal tract and thereby provide a sustained, i.e.,extended or prolonged, action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. They may also be coated using the techniques described inthe U.S. Pat. Nos. 4,256,108, 4,166,452 and 4,265,874 to form osmotictherapeutic tablets for controlled release.

The pharmaceutical compositions of the invention may be formulated forcontrolled release of the active agent. Such compositions may beformulated as controlled-release matrix, e.g., as controlled-releasematrix tablets in which the release of a soluble active agent iscontrolled by having the active diffuse through a gel formed after theswelling of a hydrophilic polymer brought into contact with dissolvingliquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymershave been described as capable of forming such gel, e.g., derivatives ofcellulose, in particular the cellulose ethers such as hydroxypropylcellulose, hydroxymethyl cellulose, methylcellulose or methylhydroxypropyl cellulose, and among the different commercial grades ofthese ethers are those showing fairly high viscosity. In otherconfigurations, the compositions comprise the active agent formulatedfor controlled release in microencapsulated dosage form, in which smalldroplets of the active agent are surrounded by a coating or a membraneto form particles in the range of a few micrometers to a fewmillimeters.

Another contemplated formulations are depot systems, based onbiodegradable polymers, wherein as the polymer degrades, the activeagent is slowly released. The most common class of biodegradablepolymers is the hydrolytically labile polyesters prepared from lacticacid, glycolic acid, or combinations of these two molecules. Polymersprepared from these individual monomers include poly (D,L-lactide)(PLA), poly (glycolide) (PGA), and the copolymer poly(D,L-lactide-co-glycolide) (PLG).

The pharmaceutical formulations of the present invention are useful intreatment of any medical indication in which administration of 9CBC or aderivative thereof as defined herein is useful, e.g., neuro-retinaldegeneration such as age related macular degeneration (AMD) andretinitis pigmentosa, and can be provided in a variety of dosages.

The dosage will depend on the state of the patient, i.e., the subject orindividual treated, and will be determined as deemed appropriate by thepractitioner. Depending on the active agent dosage, the formulations ofthe invention may be administered, e.g., daily, twice daily, thricedaily or four times daily, for various duration periods, e.g., days,weeks, months, years, or decades.

Unless otherwise indicated, all numbers expressing, e.g., the weightratio between the active agent, viscosity enhancing agent, andantioxidant(s) composing the formulation of the present invention, asused in this specification, are to be understood as being modified inall instances by the term “about”. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in this specification areapproximations that may vary by up to plus or minus 10% depending uponthe desired properties to be obtained by the present invention.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Example 1. Synthesis of 9-cis-β-carotene Materials and Methods

Ethyl 3,3-dimethylacrylate was purchased from Sigma Aldrich.β-cyclocitral was purchased from Alfa Aeser. All-trans retinal andtriethyl 3-methyl-4-phosphono-2-butenoate were purchased from TorontoResearch Chemicals (TRC). Triphenylphosphonium bromide was purchasedfrom Acros Organics. α,α′-Azoisobutyronitrile (AIBN) was purchased fromMolekula.

Experimental (General)

All reactions were carried out under argon/nitrogen atmosphere. Thereactions (except for steps a and b in Scheme 1) were performed in thedark under red dim light.

As described herein and depicted in Scheme 1, 9-cis-β-carotene (9CBC)could be obtained by multi-step synthesis (8 steps). The building blocksfor the synthesis of 9CBC are β-cyclocitral and all-trans retinal. 9CBCcould be afforded by Wittig reaction of all-trans retinal and 9-cisretinyl triphenylphosphonium bromide salt (Goswami and Barua, 2003)which could be obtained from the building block, β-cyclocitral (Scheme2).

Synthesis of ethyl 4-bromo-3-methylbut-2-enoate

As depicted in Scheme 2, N-bromosuccinimide (85 mmol 15.1 g) and acatalytic amount of AIBN (0.02 g) were added to a solution of ethyl3,3-dimethylacrylate (78 mmol, 10 g) in carbon tetrachloride (128 mL)and the mixture was heated under reflux for 3 h. It was then cooled andfiltered, and the precipitate was washed with chloroform. The combinedorganic phases were washed with saturated aqueous sodium sulfite andbrine, dried (MgSO₄), and concentrated under reduced pressure to givethe (E) and (Z)-4-bromo-3-methylbut-2-enoate.4-Bromo-3-methylbut-2-enoate distilled under vacuum (3.6 mbar, 84-86°C.). HPLC analysis (C18-RP (5 μm) LiChrocart column): elution ofCH₃CN/H₂O, 60:40 to 100:0, in 12 min (flow rate 1 mL/min). Retentiontime of ethyl 4-bromo-3-methylbut-2-enoate was 7 min (purity: 96%). Thespectral data were consistent with literature (Mata and Thomas, 1995;Roder et al., 1980).

Reformatsky Reaction (Lactone Ring Formation) (U.S. Pat. No. 5,567,855)(Step a)

As depicted in Scheme 1, step a, in a 100 mL round-bottomed flask fittedwith a reflux condenser, nitrogen inlet, and addition funnel was added2.5 grams of zinc dust (38.28 mmol) and 4 mL of dry tetrahydrofuran. Theβ-cyclocitral (36.45 mmol, 5.88 mL) and ethyl4-bromo-3-methylbut-2-enoate (36.51 mmol, 5.52 mL) were added to theaddition funnel with 15 mL of dry tetrahydrofuran. The reactants wereadded dropwise following an initial addition of 1-2 mL to initiate thereaction. The rate of addition was adjusted to maintain a gentle reflux,with ca. 20-30 min for complete addition. The reaction solution was thenheated to reflux for 30 minutes. After cooling to room temperature, 15mL of saturated ammonium chloride solution was added and the solutionstirred for 30 min. The reaction solution was worked up with addition ofanother 30 mL of saturated ammonium chloride solution to a 500 mLseparatory funnel and dimethyl ether extraction (3×25 mL). The combinedorganic layers were washed with brine and dried over MgSO₄.Concentration in vacuo produced 8.95 g. The product was dissolved in hothexane and allowed to recrystallize with cooling. The white solid wasfiltered and collected to yield 4.68 g lactone (55%). ¹H-NMR (CDCl₃, 400MHz); d (ppm) 5.82 (s, 1H), 4.96 (dd, 13.4, 4.4 1H), 2.88 (dd, 13.4,18.5, 1H), 2.16 (dd, 18.5, 3.4, 1H), 1.98 (s, 3H), 1.96 (m, 1H), 1.75(s, 3H), 1.57-1.65 (m, 2H), 1.42-1.50 (m, 2H), 1.09 (s, 3H), 0.97 (s,3H). MS (ES⁺) cacld for C₁₅H₂₂O₂ 234.3, found 235.3 (M-H+). HPLCanalysis (C18-RP (5 am) LiChrocart column): elution of CH₃CN/H₂O, 60:40to 100:0, in 12 min (flow rate 1 mL/min, λ=241 nm). Retention time oflactone was 9 min (purity: 96%).

Lactone Reduction (U.S. Pat. No. 5,567,855) (Step b)

As depicted in Scheme 1, step b, to 24 mL of tetrahydrofuran (THF) wasadded the lactone (14.08 mmol, 3.3 g). The resulting solution was cooledin a −78° C. bath and with constant stirring was aided DIBAL (18.4 mL,1.0 M in CH₂Cl₂) dropwise, never allowing the solution to warm above−40° C. (2 h addition time). After 15-20 minutes the reaction wascomplete, as evidenced by TLC (Et₂O/Hexane; 40:60). To the cooledsolution was added 500 mL of 10% H₂O/THF as a quenching solution. Thesolution was removed from the bath and allowed to warm to roomtemperature, while monitoring the rate of warming. After reaching ca.10°−15° C., then was cooled by using of the cooling bath again. At thispoint, 2 g of Na₂SO₄ was added and the resulting suspension was stirredfor 0.5 hours. The solution was filtered through a bed of celite, thebed washed with THF and the solution concentrated to yield oil withsolid ppt (2.76 g). According to MS (ES⁺) the aldehyde (step c) wasformed with mass of 219.3 (M-H+).

Ring Opening of Lactol (Aldehyde Formation) (U.S. Pat. No. 5,567,855)(Step c)

As depicted in Scheme 1, step c, In order to complete aldehyde formationstep c was performed. To a solution of 23 mL of 1,2-dichloroethane wasadded 2.76 g of the lactol and 11.5 mL of 1N HCl solution. The two phasesolution was heated to 40° C., and monitored by TLC. The solution turnedorange after 30 min. The reaction was found to be complete after 12.5hours (formed orange-red solution). The reaction was quenched by theseparation of layers and 50 mL of saturated Na₂CO₃ carefully added tothe organic layer with stirring. The aqueous layer was washed with twoportions of dichloromethane and the collective organic layers washedonce with brine. The organic solution was then dried over K₂CO₃(anhydrous) and concentrated in vacuo to afford a red-orange oil toafford 2.274 grams (60% for steps b and c). ¹H-NMR (CDCl₃, 400 MHz): d(ppm) 10.16 (d, 8.0, 1H), 7.08 (d, 16.0, 1H), 6.63 (d, 15.0, 1H), 5.86(d, 8.0, 1H), 2.12 (s, 3H), 2.05 (t, 6.0, 2H), 1.75 (s, 3H), 1.57-1.75(m, 2H), 1.40-1.53 (m, 2H), 1.05 (s, 6H). MS (ES⁺) cacld for C₁₅H₂₂O218.3, found 219.3 (M-H⁺). HPLC analysis (C18-RP (5 μm) LiChrocartcolumn): elution of CH₃CN/H₂O, 60:40 to 100:0, in 12 min and then 100%CH₃CN for 5 min (flow rate 1 mL/min, λ=396 nm). Retention time oflactone was 13 min (purity: 90%).

Horner-Emmons Reaction (9-Cis Retinyl Ester Formation) (U.S. Pat. No.5,567,855; Bennani and Boehm, 1995) (Step d)

As depicted in Scheme 1, step d, a solution of triethyl3-methyl-4-phosphono-2-butenoate (14.42 mmol, 3.51 mL) in anhydrous THF(21.7 mL) was cooled to 0° C. and treated with anhydrous DMPU (29.7mmol, 3.57 mL) and n-BuLi in hexanes (6.08 mL of 2.5 M solution, 15.21mmol). The mixture was stirred at this temperature for 20 min and thencooled to −78° C. A solution of aldehyde (7.93 mmol 1.73 g) in THF (21.7mL) was slowly added and the reaction mixture stirred at −78° C. for anadditional 60 min. The mixture was allowed to warm to 0° C. to effectcompletion of the reaction (TLC, 8:2 hexane:diethyl ether). A saturatedsolution of ammonium chloride (15 mL) was added and the mixtureextracted with EtOAc (3×20 mL). The organic layer was washed with water(2×10 mL) and brine (20 mL), dried over MgSO₄, and concentrated to give6.33 g red oil. The residue was purified on a short SilicaGel column(95:5 hexane: diethyl ether) to give 1.85 g (71% yield) of the desiredester. 9-cis retinyl ester from its trans isomer by preparative HPLC(cosmosil cholester packed column 20.0 mm I.D×250 mm) using elution ofCH₃OH/H2O, 90:10 for 4 min then, 90:10 to 95:05 for 4 min, and then95:05 for 17 min (flow rate 25 mL/min, λ=402 nm). Retention time was13.7 min.

Analytic HPLC analysis (cosmosil cholester packed column 4.6 mm I.D×250mm): isocratic elution of CH₃OH/H₂O 95:05, in 12 min (flow rate 2mL/min). Retention time was 8 min (99.6% purity). ¹H-NMR (CDCl₃, 400MHz); d (ppm) 7.08 (dd, 15, 11.3, 1H), 6.65 (d, 16, 1H), 6.29 (d, 15,1H), 6.22 (d, 15, 1H), 6.05 (d, 11.9, 1H), 5.77 (s, 1H), 4.17 (q, 2H),2.34 (s, 3H), 2.05 (t, 2H), 2.00 (m, 3H). 1.75 (s, 3H), 1.64 (m, 2H),1.49 (m, 2H), 1.28 (t, 3H), 104 (s, 6H). MS (ES⁺) cacld for C₂₂H₃₂O₂328.5, found 329.4 (M-H+). 2D COSY NMR experiment (700 MHz) proved that9-cis retinyl ester was obtained.

Ester Reduction (9-cis-retinol Formation) (Step e) (Alvarez et al.,2001)

As depicted in Scheme 1, step e, to a solution of ethyl 9-cis retinylester (1.674 mmol 0.55 g) in THF (5 mL) at −78° C., was added DIBAL-H (7mL, 1M in hexane, 7 mmol) and the resulting suspension was stirred for 1h at −78° C. After careful addition of 10% aqueous NH₄Cl, the mixturewas extracted with ethyl ether (3×0 mL). The combined organic layerswere dried over MgSO₄ and concentrated in vacuo. The residue was used inthe next step without further purification. Analytic HPLC analysis(cosmosil cholester packed column 4.6 mm I.D×250 mm): Elution ofCH₃CN/H₂O, 65:35 to 80:20, in 20 min, and then 100% CH₃CN for 8 min(flow rate 2 mL/min, λ=370 nm). ¹H-NMR (400 MHz) showed typical peaks ofthe alcohol group at 4.25 ppm (2H) and 3.71 ppm (OH, broad peak).Retention time was 13.4 min (87% purity). MS (ES⁺) cacld for C₂₀H₃₀O286.45, found 269.3 (M-(OH)).

9-cis-retinylphosphonium Salt Formation 1 (Step f)

As depicted in Scheme 1, step f, to a solution of 9-cis retinol (0.77mmol, 220 mg) in dry MeOH (1 ml), triphenylphosphine hydrobromide (0.88mmol 320 mg) in MeOH was added dropwise with stirring. The mixture wasstirred at room temperature under argon. After 1 h, the solvent wasevaporated in a rotary evaporator and the light orange residue waswashed with 10 ml portions of hexane 5-6 times to remove side products.Analytic HPLC analysis (cosmosil cholester packed column 4.6 mm I.D×250mm): Elution of CH₃CN (0.1% TFA)/H₂O (0.1% TFA), 65:35 for 4 min, thenfrom 65:35 to 75:25 for 8 min, and finally isocratic elution 75:25 CH₃CN(0.1% TFA)/H₂O (0.1% TFA) for 4 min (flow rate 2 mL/min, λ=370 nm).Retention time was 10.8 min (72% purity). MS (ES⁺) cacld for C₃₈H₄₄P⁺531.3, found 531.4 (M), 532.3 (M-H+).

Wittig Reaction 9CBC Formation (Step g) (Goswami and Barua, 2003)

As depicted in Scheme 1, step g, to a stirred solution of 86 mg of 9-cisretinyl triphenylphosphonium bromide (0.14 mmol) in 2 ml of dry ethanolat 70° C., all-trans retinal (19.7 mg, 0.069 mmol) in 17 ml of dryethanol was added dropwise. KOH (0.43 g) dissolved in dry ethanol (5 ml)was added slowly to the mixture, and the solution was stirred at 70° C.for 15 min and then at room temperature for 2 h. The reaction wasmonitored by TLC (8:2 hexane:ethyl acetate). When no more caroteneformation was noted after 2 h, water (10 ml) was added to the reactionmixture and the product was extracted with portions of diethyl ether.The organic phase was removed under vacuum. MS (APPI) of crude cacld forC₄₀H₅₆ 536.4, found 536.4 (M), 537.4 (M-H+). HPLC analysis of the crude(cosmosil cholester packed column 4.6 mm I.D×250 mm) isocratic elutionof CH₃OH/CHCl₃, 80:20 for 16 min (flow rate 2 mL/min, λ=447 nm).Retention time was 11 min. (87% purity, FIG. 1). 9CBC purified bypreparative HPLC (Cholester packed column) using elution of CH₃OH:CHCl₃,85:15 for 5 min, then 85:15 to 80:20 for 5 min and then isocraticelution of 80:20 for 20 min (flow rate 25 mL/min, λ=447 nm). Retentiontime was 16 min. MS (APPI) of crude cacld for C₄₀H₅₆ 536.4, found 536.4(M), 537.4 (M-H⁺).

It is also possible to substitute CHCl₃ with methylene chloride forpreventing possible damage during separation. Likewise, the finalseparation can be done with Methyl tert-butyl ether and acetonitrile,instead of the solvents mentioned above.

Example 2. Stable Formulations of 9-cis-β-carotene

Purified 9CBC was found to be unstable under vacuum or nitrogenflushing. In this study, various 9CBC formulations comprising a naturalor synthetic commercially available antioxidant(s), a viscosityenhancing agent, or both, were prepared, and the chemical stability ofsome of these formulations were tested.

The specific formulations prepared were:

-   -   1. Lecithin+antioxidants mix [a pellet content from the        commercially available product CarotenALL, Mixed Carotenoid        Complex (Jarrow Formulas)]+9CBC (10:5:1 w/w/w)    -   2. Lecithin+BHT+9CBC (10:5:1 w/w/w)    -   3. Miglyol 810+antioxidant mix+9CBC (100:5:1)    -   4. Miglyol 810+BHT+9CBC (100:5:1)    -   5. Tween 40+9CBC    -   6. Tween 20+antioxidant mix+9CBC    -   7. Tween 40+antioxidant mix+9CBC

Table 1 shows spectral tests confirming the stability of 9CBC in theformulations tested within the specified time period. As clearlyindicated, lecithin as an excipient and binder plays a crucial role instabilizing 9-CBC. Interestingly, although a formulation comprising anantioxidant only as the anti-degradative measure (as disclosed in U.S.Pat. No. 5,612,485) has somewhat of a positive effect during the first14 days, said formulation has rapidly degraded during the next days,wherein the measurable 9-CBC by day 22 was almost as low as in the blankformulation. In fact, as shown in Table 1, the amount of 9CBC in saidantioxidant-based formulation was significantly lower than that in theformulation that further contained a viscosity enhancing agent(lecithin), already after 14 days, suggesting that 9CBC degradationstarts during the first 14 days and intensifies later. In sharpcontrast, the measurable amount of 9CBC in the formulation containingboth antioxidant and a viscosity enhancing agent was almost 100%compared to the initial amount, indicating the stability of saidformulation.

TABLE 1 Results of spectral tests of the various formulations testedAbsorption at Absorption at 447/450 nm 447/450 nm Tested medium 14 days22 days Diethyl ether 0 0 Diethyl ether + lecithin + BHT 0.71/0.690.73/0.70 Diethyl ether + BHT + 9CBC 2.11/2.08 1.05/0.96 Diethyl ether +lecithin + BHT + 2.43/2.32 2.42/2.30 9CBC

REFERENCES

-   Alvarez, R.; Dominguez, B.; de Lera, A. R., Synth. Commun. 2001, 31,    2083-2087-   Alvarez, R.; Domínguez, M.; Pazos, Y.; Sussman, F.; de Lera, A. R.,    Chem. Eur. J. 2003, 9, 5821-5831-   Bennani, Y. L.; Boehm, M. F. J. Org. Chem. 1995, 60, 1195-1200-   Goswami, B. C.; Barua, A. B., J. Labelled Comp. Radiopharm. 2003,    46, 843-849-   López, S.; Montenegro, J.; Saá, C., J. Org. Chem. 2007, 72,    9572-9581-   Mata, E. G.; Thomas, E. J., J. Chem. Soc.-Perkin Trans. 1 1995,    785-799-   Roder, E.; Wiedenfeld, H.; Frisse, M., Arch. Pharm. 1980, 313,    803-806

1. A formulation comprising an active agent, a thickening/solidifyingagent, and an antioxidant, wherein said active agent is 9-cis-β-carotene(9CBC) or a derivative thereof of the formula C:

wherein R₂ is H or methyl; X is (—C═C—C—)_(n) optionally substitutedwith one or more methyl groups; and n is an integer of 0-16.
 2. Theformulation of claim 1, wherein said thickening/solidifying agentcomprises a lecithin or a PEGylated derivative thereof; a lecithin-likesubstance; a medium chain triglyceride or a mixture thereof; a povidone;a polysorbate; or sorbitol.
 3. The formulation of claim 2, wherein saidthickening/solidifying agent comprises a lecithin or a PEGylatedderivative thereof, a lecithin-based product, or a povidone.
 4. Theformulation of claim 3, wherein said lecithin is egg lecithin or soybeanlecithin; or said lecithin-based product is L-α-lecithin, granular(Acros Organics™), Phospholipon® 50, Phospholipon® 75, Phospholipon®85G, Phospholipon® 90G, Phospholipon® 80H, Phospholipon® 90H,Phospholipon® E25, Phospholipon® E35, Phospholipon® E, Phospholipon®LPC20, Phospholipon® LPC25, or Phospholipon® LPC65.
 5. The formulationof claim 2, wherein said lecithin-like substance is egg yolk; saidmedium chain triglyceride is miglyol, miglyol 810, or a mixture thereof;or said polysorbate is polysorbate 20, polysorbate 40, polysorbate 60,or polysorbate
 80. 6. The formulation of claim 1, wherein saidantioxidant is a thiol; a sulphoximine; a metal chelator; sodiumbisulfite; sodium metabisulfite; a vitamin; a phenol; a benzoate; uricacid; mannose; propyl gallate; a selenium; a stilbene; a carotenoid; ora mixture thereof.
 7. The formulation of claim 6, wherein saidantioxidant is a phenol selected from the group consisting of BHT,butylhydroxyanisole, ubiquinol, nordihydroguaiaretic acid, andtrihydroxybutyrophenone; a carotenoid selected from the group consistingof α-carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin,astaxanthin, and lycopene; or a mixture thereof.
 8. The formulation ofclaim 7, wherein said antioxidant is BHT, a carotenoid, or a mixturethereof.
 9. The formulation of claim 1, wherein saidthickening/solidifying agent comprises a lecithin or a PEGylatedderivative thereof, a lecithin-based product, or a povidone; and saidantioxidant is BHT, butylhydroxyanisole, ubiquinol, nordihydroguaiareticacid, trihydroxybutyrophenone, α-carotene, β-carotene, β-cryptoxanthin,lutein, zeaxanthin, astaxanthin, lycopene, or a mixture thereof.
 10. Theformulation of claim 9, wherein said lecithin is egg lecithin or soybeanlecithin; said lecithin-based product is L-α-lecithin, granular (AcrosOrganics™), Phospholipon® 50, Phospholipon® 75, Phospholipon® 85G,Phospholipon® 90G, Phospholipon® 80H, Phospholipon® 90H, Phospholipon®E25, Phospholipon® E35, Phospholipon® E, Phospholipon® LPC20,Phospholipon® LPC25, or Phospholipon® LPC65; or said antioxidant is BHT,a carotenoid, or a mixture thereof.
 11. The formulation of claim 9,wherein said thickening/solidifying agent comprises egg lecithin orsoybean lecithin; and said antioxidant is BHT, α-carotene, β-carotene,β-cryptoxanthin, lutein, zeaxanthin, astaxanthin, lycopene, or a mixturethereof.
 12. The formulation of claim 11, wherein saidthickening/solidifying agent comprises egg lecithin or soybean lecithin;and said antioxidant is BHT.
 13. The formulation of claim 9, wherein theweight ratio between said thickening/solidifying agent and saidantioxidant or mixture thereof in said formulation is in a range of100:1 to 1:100, respectively.
 14. The formulation of claim 1, whereinsaid active agent is 9CBC.
 15. The formulation of claim 14, comprising9CBC, a soybean lecithin and BHT, at a weight ratio of about 1:10:5,respectively.
 16. The formulation of claim 1, which is chemically stablefor at least 72 hours under inert conditions.
 17. The formulation ofclaim 1, in the form of a solid, semi-solid, gel, or paste.
 18. Theformulation of claim 17, formulated for oral administration. 19.(canceled)
 20. A method for the synthesis of 9-cis β-carotene (9CBC) ora derivative thereof, said method comprising: (i) reducing4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-one,herein identified compound 3, and opening the ring of the lactolobtained with complete retention of the double bond configuration, tothereby obtain(2Z,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-2,4-dienal,herein identified compound 5; (ii) subjecting the(2Z,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-2,4-dienalto Horner-Emmons reaction to obtain a 9-cis retinyl ester, hereinidentified compound 6; (iii) reducing the 9-cis retinyl ester to obtain9-cis retinol, herein identified compound 7; and (iv) converting the9-cis retinol to its triphenylphosphonium salt, herein identifiedcompound 8, and subjecting said triphenylphosphonium salt to Wittigreaction with a compound of the formula A:

wherein R₂ is H or methyl; X is (—C═C—C—)_(n) optionally substitutedwith one or more methyl groups; and n is an integer of 0-16, in thepresence of a strong base, to thereby obtain said 9CBC or derivativethereof.
 21. The method of claim 20, wherein: (i) the reduction in step(i) is carried out with diisobutylaluminum hydride (DIBAL-H); or (ii)opening the ring of the lactol in step (i) is carried out in thepresence of a strong acid; or (iii) the Horner-Emmons reaction in step(ii) is carried out with triethyl 3-methyl-4-phosphono-2-butenoate toobtain 9-cis retinyl ethyl ester; or (iv) converting the 9-cis retinolto its triphenylphosphonium salt in step (iv) is carried out withtriphenylphosphine; or (v) said compound of the formula A is all-transretinal, to obtain 9CBC. 22-25. (canceled)
 26. The method of any one ofclaim 20, wherein said4-methyl-6-(2,6,6-trimethylcyclohex-1-en-1-yl)-5,6-dihydro-2H-pyran-2-oneis synthesized by a method carried out as depicted in Scheme 1 andcomprising reacting β-cyclocitral, herein identified compound 1, with acompound of the formula B:

wherein R₁ is (C₁-C₈)alkyl or (C₆-C₁₀)aryl, in the presence of metallicZn, in a Reformatsky reaction.
 27. The method of claim 26, wherein saidβ-cyclocitral is reacted with a compound of the formula B wherein R₁ isethyl.
 28. 9-cis-β-carotene (9CBC) or a derivative thereof, synthesizedaccording to the method of claim 20, wherein said derivative is of theformula C:

wherein R₂ is H or methyl; X is (—C═C—C—)_(n) optionally substitutedwith one or more methyl groups; and n is an integer of 0-16.
 29. Theformulation of claim 6, wherein said thiol is selected from the groupconsisting of aurothioglucose, dihydrolipoic acid, propylthiouracil,thioredoxin, glutathione (GSH), L-cysteine, N-acetylcysteine (NAC),cystine, cystamine, and thiodipropionic acid; said sulphoximine isselected from the group consisting of buthionine-sulphoximine,homo-cysteine-sulphoximine, buthionine-sulphone, and penta-, hexa- orheptathionine-sulphoximine; said metal chelator is selected from thegroup consisting of a α-hydroxy-fatty acid, palmitic acid, phytic acid,lactoferrin, citric acid, lactic acid, malic acid, humic acid, bileacid, bile extract, bilirubin, biliverdin, EDTA, Na₂-EDTA, Na₂-EDTA-Ca,EGTA, and DTPA; said vitamin is selected from the group consisting ofvitamin E, ascorbic acid, and salts thereof; said phenol is selectedfrom the group consisting of butylhydroxytoluene (BHT),butylhydroxyanisole, ubiquinol, nordihydroguaiaretic acid, andtrihydroxybutyrophenone; said benzoate is coniferyl benzoate; saidselenium is selenium-methionine; said stilbene is selected from thegroup consisting of stilbene oxide, and trans-stilbene oxide; and saidcarotenoid is selected from the group consisting of α-carotene,β-carotene, β-cryptoxanthin, lutein, zeaxanthin, astaxanthin, andlycopene.